I. Field of the Invention
The present invention relates to the expression of exogenous proteins in plants. In particular, this invention relates to the use of downstream elements, specifically introns, to enhance expression of certain desired exogenous proteins in plants. Additionally, this invention relates to the use of methods to enhance expression of proteins of pharmacological interest in food plants. Methods and compositions for the enhancement of protein expression in plants and plant cells are disclosed.
II. Description of Related Art
The amount of protein that is synthesized from a gene is a function of several complex and interacting processes. Transcription, RNA maturation, and translation are each comprised of a large number of events, all of which are potentially regulated either independently or in concert. It is widely recognized that the upstream elements that control transcription and translation have very significant roles in determining the quantity, timing, and tissue specificity of gene expression. However, the sequences that are required for other aspects of expression (such as RNA processing) could be of equal or greater importance, and might be located in virtually any part of a gene.
While an enormous amount of research has demonstrated the importance of 5'elements in controlling gene expression, evidence shows that sequences downstream of the start of transcription can also have a major influence on the level or pattern of expression of plant genes. These elements include protein coding sequences (De Almeida et al., 1989; Douglas et al., 1991), introns (Callis et al., 1987; Clancy et al., 1994), and 5' or 3' transcribed but untranslated sequences (Dean et al., 1989; Larkin et al., 1993; Ulsamov and Folk, 1995). Despite the documented importance of downstream elements on gene expression, these regions are included in only a minority of reporter gene fusions designed to investigate the expression pattern of a gene.
The use of intronic portions of genes to aid in gene expression has also been reported for certain genes. For example, Callis et al. reported that at least the first intron of the maize (Zea mays L.) alcohol dehydrogenase -1 (Adh1) gene was probably required for significant levels of expression. (Callis et al., 1987). Further, Callis et al. reported that the position of the first intron with respect to the chimeric chloramphenicol acetyl transferase (cat) gene to be expressed was important to the level of expression observed. Illustrative of this point are the pACI.sub.1 I.sub.8,9 A and pAI.sub.1 CI.sub.8,9 A constructs wherein A represents Adh1, I.sub.X represents intron number x and C represents the cat gene. The construct wherein intron 1 was located upstream from the CAT gene showed a 110-fold increase in transient gene expression relative to pACI.sub.8,9 A, but a 21-fold increase relative to pACI.sub.1 I.sub.8,9 A (i.e., 110-fold relative to a construct lacking the first intron, but 21-fold relative to one in which the first intron is downstream of the cat gene).
It has also been shown that compositions comprising the Zea mays L. (maize) Sh1 first intron fused to short sections of the flanking exons increases transient reporter gene expression in protoplasts of several grass species (Vasil et al., 1989). Further, Clancy et al. (1994) showed that in some instance, variations can be made in the intron structure and length without affecting the rate of enhancement of protein expression. Nevertheless, it is also known that not all genes containing introns require the presence of introns for efficient expression. For example, Chee et al., (1986) showed that removal of introns from bean phaseolin genes transferred to tobacco did not affect the expression of phaseolin protein in callus tissue.
The histochemical staining patterns previously reported for intronless GUS fusions to the tryptophan pathway genes TSB1, TSB2, ASA1, and ASA2 are similar to the patterns seen in lines containing an intronless GUS fusion to the tryptophan pathway gene PAT1 (Niyogi, 1993; Pruitt and Last, 1993). In light of the poor expression of PAT1-GUS in the absence of an intron detailed below, it seems plausible that these fusions might under-represent the expression of the genes under study. Consistent with this hypothesis, results analogous to those reported for PAT1-GUS fusions with and without introns were obtained for all three of the Arabidopsis genes encoding the next tryptophan pathway enzyme, phosphoribosylanthranilate isomerase (J. Li and R. L. Last, unpublished data). In each case, GUS fusions containing introns and the transit peptide give much more intense and widespread histochemical staining in transgenic plants than fusions lacking introns and the transit peptide.